L15/16 Eukaryotic Translation Flashcards
Translation eukaryotes vs prokaryotes
Eukaryotes:
- transcription and translation not coupled
- internal translation initiation within mRNA does not usually occur in eukaryotes
- monocistronic mRNA
- mechanics very similar to prokaryotes
- in eukaryotes, 5’ cap essential to beginning translation
Ribosome
- ribosomes synthesized in electron dense nucleolus
- transported to cytoplasm
- nucleolus attached to nuclear membrane
Translation mechanism
See onenote diagram
Translation initiation codon
- most translational initiation occurs at the first AUG, scanned from 5’ cap of mRNA
- efficiency is influenced by fit to a consensus (Kozak consensus)
The closer the sequence is to the consensus, the better the translation initiation sequence the AUG is
E.g. 90% uses the nicer sequence, 10% uses the less fitting sequence - gives us different length proteins
- downstream AUG may be used in some cases
Rare instances of dicistronic mRNA
See onenote diagram
Dicistronic = mRNA that encodes two distinct proteins
Sequence between upstream termination sequence and downstream initiator sequence = IRES
E1F4F binding to IRES recruits small ribosomal subunit to begin translation
IRES = internal ribosomal entry site
mRNA translatability
See onenote diagram
- 5’ cap and polyA tail
- polyA tail determines if mRNA will be translated
- decapping or shortening polyA tail precipitates degradation of mRNA
5’ cap added for stability and translatability
E1F4E binds to 5’ cap to recruit small ribosomal subunit
Why is it a circle?
- After the ribosome falls off, easier to reinitiate translation if 5' end is near 3' end - Makes translation much more efficient, can efficiently continue rounds and rounds of translation
mRNA translatability - polyA tail
- polyA tail determines if mRNA will be translated
- The longer the poly A tail, the longer this association will last, keeps it stable
- Short poly A tails, less stable, less translation
If not translated much, usually degraded
mRNA translatability - nonsense mediated decay (NMD)
- an RNA surveillance mechanism
- directs degradation of mRNAs containing premature stop codons
- prevents production of truncated gene products
mRNA stability
See onenote diagram
- ribosomes displace exon junction complexes on mRNA
When eukaryotic RNA are processed, the removal of introns leaves protein complexes at the newly formed exon junctions - called exon junction complexes (EJC). Upon translation, the ribosome displaces the EJCs. The mRNA is then stable to be translated.
mRNA stability - premature termination due to nonsense mutation
See onenote diagram
- ribosomes do not displace complexes
- trigger RNA degradation
Checking if the mRNA is working properly:
- Pause of ribosome at premature stop codon => interaction between exon junction complex and Upf protein, causes ribosome to fall off
- Decapped, mRNA no longer stable, gets degraded
- The closer it is to the 5’ end, the more likely that the premature stop codon will be detected
Nonsense mediated decayIf there is a premature termination codon upstream (5’) of an EJC, the EJC participates in the recruitment of Upf proteins to the stalled ribosome. The Upf proteins then recruit a decapping enzyme to remove the 5’ cap and a deadenylating enzyme to remove the poly-A tail. The RNA is then rapidly degraded.
mRNA stability - premature termination due to abnormal splicing
See onenote diagram
- This mechanism looks for both mutations and improper splicing
- Upf triggers mRNA degradation
mRNA stability - non-stop mediated decay
See onenote diagram
- ribosome translates polyA tail (lysine) and stalls at the end
- mRNA lost its termination codon
- Ribosome starts translating poly A tail, AAA = lysine
- Lysine recognised, ribosome begins to stall as the tRNA with the anticodon UUU will be depleted
- Protease will degrade the polylysine, lysine is positively charged, will have a significant effect on the protein
If there is a mutation of the normal termination codon the ribosome will continue to translate through the poly-A tail (resulting in a poly-lysine polypeptide. The ribosome stalls due to depletion of lysine-charged tRNAs or reaching the end of the template.Dom34/Hbs1 recognise the stalled ribosome, Hbs1 hydrolyses GTP and leaves the complex. This allows Dom34 with the Rli1 ATPase to disassemble the ribosome and recruit an endonuclease to cut the RNA upstream of the ribosome. Exonucleases then degrade the RNA fragments.
mRNA stability - no-go mediated decay
See NMD, NSD, NGD page on onenote
If a ribosome is stalled due to RNA secondary structure or depletion of appropriately charged tRNAs, this also leads to the recruitment of Dom34/Hbs1 which recognise the stalled ribosome as for non-stop mediated decay. Hbs1 hydrolyses GTP and leaves the complex. This allows Dom34 with the Rli1 ATPase to disassemble the ribosome and recruit an endonuclease to cut the RNA upstream of the ribosome. Exonucleases then degrade the RNA fragments
Programmed frameshifting
See onenote diagram
- retroviruses
Post-translation
See onenote
- polypeptide maturation
- chaperones assist correct folding
- folding, co-factor binding, interaction with other polypeptides
- incorrectly folded and aberrant proteins are degraded
